The present invention relates to chemiluminescent assays which incorporate a second film or membrane which includes a solid chemical component for activation of a stable dioxetane. Decomposition of dioxetane can be accomplished using heat and chemical treatment.
Recently a variety of non-isotopic labeling methods have been developed to replace radioactive labels in DNA probe-based assays. It is most common in such methods to use marker enzymes to detect nucleic acid probes using either colormetric, chemiluminescent, bioluminescent or fluorescent methods. Each of these methods have been used reliably for both hybridization of DNA probe-based assays for nucleic acid detection as well as in solid-phase immunochemical assays wherein the target molecule is typically an antigen of interest.
Regardless of the type of non-isotopic detection method used, the labels are measured directly with fluorophores (without use of enzymes) or indirectly using enzyme amplification schemes. Wherein the label is detected directly without an enzymatic reaction, sensitivity is generally less. Typically, in an indirect labeling scheme, a label is incorporated into the probe or the analyte in the form of a small molecule such as digoxigenin, fluorescein or biotin. This label may or may not be detectable on its own and its presence is revealed using enzyme conjugates that specifically bind to the small molecule in the probe. A clear advantage of an indirect labeling scheme is the increased sensitivity one achieves through enzymatic amplification of the signal. However, a disadvantage of such methods as they are currently practiced in the field is that many steps are required in the assay protocol, requiring more time to complete the assay. Moreover, a greater number of reagents are required which means greater cost. In addition, where the method of detection is enzyme-based, stability of the enzyme and its shelf life need to be considered if one is to expect optimum performance of the assay.
Chemiluminescence detection relies on a chemical reaction that generates light. It is this method which is now widely used for both nucleic acid detection as well as solid-based immunodetection due to its high sensitivity and wide variety of analysis methods ranging from manual film reading to instrumentation for processing images. One non-radioactive detection method now commercially available is the DIG-system (Boehringer-Mannheim) which uses digoxigenin, a small molecule, as a label for a probe. After binding of a DIG-labeled probe to a target molecule, an anti-DIG antibody conjugated to the enzyme alkaline phosphatase is added. Detection is achieved through the enzymatic dephosphorylation of a 1,2-dioxetane substrate which leads to the production of a chemiluminescent signal. This method relies on an enzymatic means of amplification of the signal and as such presents a disadvantage in that considerations regarding the stability of the enzyme and its shelf life are important. In addition, several steps are required in the protocol, including the binding of a probe to an enzyme-conjugated antibody. The shelf life of an antibody is an additional consideration.
In view of the simplicity of chemical reactions relative to enzymatic reactions, it would be desirable to achieve chemiluminescent signal amplification by chemical as opposed to enzymatic means. U.S. Pat. No. 5,516,636 to McCapra and a later publication by Schubert (Nucleic Acids Research, 1995, Vol. 23, No. 22 p. 4657) describe the use of sensitizer-labeled oligonucleotide probes for the detection of nucleic acid target molecules. In a solid phase DNA probe assay, a DNA target molecule is bound to a membrane and hybridized to a sensitizer-labeled oligonucleotide complementary in sequence to the target DNA. The membrane is subsequently treated with an olefin solution. Upon exposure of the membrane to ambient oxygen and light, the sensitizer molecules become excited and transfer their excess energy to ambient oxygen for formation of singlet oxygen. The singlet oxygen therein produced reacts with the olefin on the membrane to form a stable 1,2-dioxetane in the area of the hybridization zone which when subsequently exposed to heat, chemical treatment or enzymatic treatment decomposes to emit light. Thus, oligonucleotides labeled with sensitizer are able to amplify the dioxetane concentration based on repeated excitation/oxygen quenching cycles to achieve a high level of sensitivity.
McCapra discloses that wherein the dioxetane contains a phenolic hydroxyl protecting group the triggering chemical mechanism for decomposition is the raising of pH. However, he fails to disclose particular bases suitable for the decomposition or the exact method by which it can be accomplished.
Schubert uses chemical treatment of a thermally stable dioxetane as a means of decomposing the dioxetane wherein decomposition is achieved by a change of pH using a liquid triggering solution of tetrabutyl ammonium hydroxide. While an advantage of chemical treatment includes speed and efficiency, a disadvantage of the method of Schubert lies in the use of a liquid base solution which can be caustic, inconvenient and messy to use. Schubert discloses that deprotonation via base treatment of the phenolic hydroxyl protecting group of the dioxetane used causes it to lose its thermal stability and decay with accompanying emission of light.
The prior art fails to teach the combined use of heat and chemical treatment as a means of decomposing dioxetane. It would seem therefore that there is a need for a method that could allow for the combined use of heat and chemical treatment as the triggering means for an even greater enhancement of decomposition of a stable dioxetane. Thus, if decomposition of the dioxetane acts as the bottleneck for production of a signal, it is important that the conditions under which decomposition occur be optimal. Combining chemical treatment with heat would allow for this. However, up until now it has not been possible to use both chemical treatment and heat. Heating a caustic solution of base to at or near boiling temperatures would be both dangerous and impractical.
There is therefore a need for a method of providing a chemical triggering agent in a dry form which upon exposure to an appropriate energy source such as heat can be activated for enhanced decomposition of a thermally stable dioxetane and a resultant enhancement of the chemiluminescent signal. It would be a further advantage to provide for a way to use a similar dry agent to trigger dioxetane decomposition for use in both solid-phase immunoassays and nucleic acid assays.